Cellular phenotype is commonly characterized by mRNA expression profiling. However, in heterogeneous populations of cells, profiling cell populations in bulk discards all information pertaining to the associations of specific mRNA transcripts in individual cells. In immunoglobulin-producing lymphocytes, for example, pooled sequencing results in the separate characterizations of immunoglobulin heavy- and light-chains, peptides whose co-expression within a cell determines an immunoglobulin's antigen-specificity. To resolve this, heavy- and light-chain mRNA produced by individual lymphocytes may be made to associate in the sequence of a polymerase chain reaction (PCR) product (Embleton M J, et al. Nucleic Acids Res. 1992 Aug. 11; 20(15): 3831-3837). In this method, cells from two clonal populations were fixed and permeabilized, their heavy- and light-chain mRNA reverse transcribed to cDNA, and the cDNA amplified by PCR with primers containing reverse-complementary overhangs which allowed heavy- and light-chain PCR product to cross-link during the reaction. The fidelity of chimeric heavy/light chain PCR product to the original intracellular co-localization could then be verified either using fluorescent primers using fluorescence microscopy or by screening of bacterial colonies transfected with chimeric PCR product. (Embleton M J, et al. Nucleic Acids Res. 1992 Aug. 11; 20(15): 3831-3837).
High-throughput sequencing can identify large numbers of heavy- and light-chain variable regions (VH and VL) in a given B-cell repertoire, but information about endogenous pairing of heavy and light chains is lost after bulk lysis of B-cell populations. A way to retain this pairing information involves depositing single B cells (>5×104 capacity per experiment) in a high-density microwell plate (125 pl/well) and lysing (DeKosky B J, et al. Nat Biotechnol. 2013 February; 31(2):166-9). mRNA is then captured on magnetic beads, reverse transcribed and amplified by emulsion VH:VL linkage PCR and the linked transcripts are analyzed by Illumina high-throughput sequencing (DeKosky B J, et al. Nat Biotechnol. 2013 February; 31(2):166-9).
RNA sequencing (RNA-Seq) is a powerful tool for transcriptome profiling, but is hampered by sequence-dependent bias and inaccuracy at low copy numbers intrinsic to exponential PCR amplification. To mitigate these complications to allow truly digital RNA-Seq, a large set of barcode sequences is added in excess, and nearly every cDNA molecule is uniquely labeled by random attachment of barcode sequences to both ends (Shiroguchi K, et al. Proc Natl Acad Sci USA. 2012 Jan. 24; 109(4):1347-52). After PCR, paired-end deep sequencing is applied to read the two barcodes and cDNA sequences. Rather than counting the number of reads, RNA abundance is measured based on the number of unique barcode sequences observed for a given cDNA sequence (Shiroguchi K, et al. Proc Natl Acad Sci USA. 2012 Jan. 24; 109(4):1347-52). The barcodes may be optimized to be unambiguously identifiable, even in the presence of multiple sequencing errors. This method allows counting with single-copy resolution despite sequence-dependent bias and PCR-amplification noise, and is analogous to digital PCR but amendable to quantifying a whole transcriptome (Shiroguchi K, et al. Proc Natl Acad Sci USA. 2012 Jan. 24; 109(4):1347-52).
Prior technologies, such as those described above, to identify the contents of individual cells required their dissociation so that they may be analyzed individually. In the case of structurally fragile cell types, such as neurons, this generally required severing whole cell parts, such as dendrites that branch from the main cell body.
Fluorescence in situ sequencing, or FISSEQ, is a method that allows the acquisition of mRNA/cDNA sequences directly from within cell monolayers or whole mount embryos (Lee J H et al. Science. 2014 Mar. 21; 343(6177):1360-3). mRNA transcripts are reverse-transcribed into cDNA and fixed to the cellular matrix. mRNA is degraded, and the cDNA subsequently circularized so that polonies comprising long repeats of cDNA sequence may be formed by rolling circle amplification (RCA). SOLiD sequencing (sequencing by oligonucleotide ligation and detection) is then used to read out 30 bp reads that allow comparison with reference gene transcripts. In order to resolve individual transcripts, signals are suppressed so that polonies are sufficiently sparse to be distinguished from one another optically. FISSEQ requires high-quality optics for each sample to be analyzed. Moreover, because of trade-offs between a microscope's depth-of-field and its imaging resolution, samples must be properly arranged on a two-dimensional plane to be analyzed.
Citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention.